Method for selecting genetically transformed cells

ABSTRACT

An environmental friendly and non toxic method for selection of transformed cells from a population consisting of transformed and non-transformed cells. The method comprises the following steps a) introducing into a cell at least one desired nucleotide sequence and at least one selection-nucleotide sequence to obtain a genetically transformed cell, wherein the selection-nucleotide sequence comprises a region which codes for a protein involved in the metabolizing of trehalose; b) placing a population with transformed and non-transformed cells into contact with trehalose and/or derivative thereof; and c) selecting the transformed cells from the population on the basis of the capacity of the transformed cells to metabolize the trehalose and/or derivative.

FIELD OF THE INVENTION

The present invention relates to a method for selecting geneticallytransformed cells.

BACKGROUND OF THE INVENTION

It is known that when genetic material is introduced into a populationof cells by means of transformation, only a number of the cells will betransformed successfully. After transformation the transformed cellsmust be identified and selected from a population of transformed andnon-transformed cells. A selection gene is therefore generally alsointroduced into the cell for this purpose in addition to the desiredtransgene. The selection gene herein codes for instance for a propertywith which the genetically transformed cells can be identified. Examplesof such selection genes are for instance genes which code for resistanceto antibiotics or herbicides. After the transformation the population oftransformed and non-transformed cells is brought into contact with theantibiotic or herbicide toxic for the non-transformed (“wild-type”)cells, so that only the transformed cells are able to survive and growdue to the presence of the introduced selection gene.

The use of such selection genes which code for antibiotic- orherbicide-resistance is however not generally desirable for transgeniccrops which are introduced on a large scale into the environment, andparticularly in food crops. Another drawback of such a selectionmechanism is further for instance that the non-transformed cells willgenerally die off, and moreover that when the population of cells is acoherent tissue of cells or a whole organism, the transformed cells canalso die as a result of for instance harmful compounds secreted by thedying, non-transformed cells.

The object of the present invention is to provide a method for selectingtransformed cells from a population of transformed and non-transformedcells, wherein the above stated drawbacks are obviated.

BRIEF DESCRIPTION OF THE INVENTION

This object is achieved with the invention by providing a methodcomprising of:

-   -   a) introducing into a cell at least one desired nucleotide        sequence and at least one selection-nucleotide sequence to        obtain a genetically transformed cell, wherein the        selection-nucleotide sequence comprises a region which codes for        a protein involved in the metabolizing of trehalose;    -   b) placing a population with transformed and non-transformed        cells into contact with trehalose and/or derivative thereof; and    -   c) selecting the transformed cells from the population on the        basis of the capacity of the transformed cells to metabolize the        trehalose and/or derivative.

DETAILED DESCRIPTION OF THE INVENTION

Trehalose is het α-1,1-disaccharide of glucose which is produced by manyorganisms, including bacteria, yeasts and fungi, as well as severalhigher plants. It is the most important blood sugar in insects.Trehalose is increasingly being used for among other things thepreparation of vaccines and in organ transplant protocols because itprovides protection against protein denaturation and membrane damage.

Cells, in particular plant cells, can not normally develop in a mediumin which an increased concentration of trehalose is present withoutanother metabolizable carbon source being present. In the methodaccording to the invention use is made hereof to select transformedcells. For this purpose the cells, in addition to using the desiredtransgene, are also transformed with a selection-nucleotide sequence,wherein the selection-nucleotide sequence comprises a region which codesfor a protein which is involved in the metabolizing of trehalose. Apopulation with transformed and non-transformed cells are then broughtinto contact with trehalose and/or a derivative thereof, for instance byadding trehalose and/or derivative thereof to the culture medium. Thetransformed cells are thus distinguished from the non-transformed cellsnot only by the relevant introduced transgene but also by the presenceof a nucleotide sequence in their genome which codes for a protein whichcan metabolize the trehalose. The transformed cells will hereby be ableto survive and grow in the medium with trehalose and/or derivative oftrehalose, while the non-transformed cells will not develop further. Inthis manner the transformed cells can thus be selected from the totalpopulation of cells on the basis of their capacity to metabolize thetrehalose and/or derivative.

The term “protein involved in the metabolizing of trehalose” relatesherein to a protein, for instance an enzyme, which is able to break downtrehalose and/or the derivative thereof, and thereby reduce theconcentration of trehalose and/or derivative.

The term “derivative of trehalose” relates to modified forms oftrehalose which can also be metabolized by the relevant protein andinduce the same response in the cells as trehalose, such as for instancemethylated or halogenated forms of trehalose.

In a preferred embodiment of the method according to the invention theintroduced selection-nucleotide sequence comprises a region which codesfor an intracellular protein with trehalase activity, i.e. an enzymeable to hydrolyze intracellular trehalose and/or derivative thereof toglucose. Owing to the presence of this protein in the transformed cells,and the absence thereof in the non-transformed cells, only thetransformed cells will be able to break down the trehalose and/orderivative which enters the cell. The intracellular concentration of thetrehalose in the transformed cells is hereby reduced, while the releasedglucose can moreover be used by the transformed cells as extra nutrientsource.

Many cells, in particular cells of higher plants, such as Glycine max.and Arabidopsis thaliana, have in their genome the gene for anendogenous trehalase (Aeschbacher R. A. et al., Plant Physiol. 119(2):489-496, 1999; Mueller et al. Plant Physiol. 125(2): 1086-1093, 2001).However, these endogenous trehalase genes generally code for anextracellular trehalase, which is not active in the cell. It is possibleto modify such endogenous genes using standard molecular biologicaltechniques. In a preferred embodiment of the method according to theinvention the selection-nucleotide sequence therefore comprises amodified endogenous trehalase gene which codes for an intracellularlyactive trehalase. The endogenous trehalase gene is herein modified suchthat the trehalase which is expressed is intracellularly active, such asfor instance by deactivating the protein-secretion signal via deletionor mutagenesis, by changing the protein targeting sequences, or the pHsensitivity of the enzymatically active site.

In a particularly suitable embodiment of the method according to theinvention the introduced selection-nucleotide sequence comprises theTreF gene from E. coli (Horlacher R. et al., J. Bacteriol. 178(1):6250-6257, 1996). This gene is simple to isolate and to introduce intodiverse cells using standard molecular biological techniques.

In another advantageous embodiment of the invention theselection-nucleotide sequence comprises the AtTRE1 gene from Arabidopsis(Locus At4G24040, AGI no. 2134960).

The method according to the invention preferably further comprises ofalso bringing the population of cells in contact with at least oneinhibitor of endogenous extracellular trehalase before or during stepb). Inhibition of the possibly present endogenous extracellulartrehalase prevents the trehalose from the medium already being partly orwholly broken down outside the cells, whereby the non-transformed cellswould also be able to develop further.

Examples of suitable inhibitors for use in the method according to theinvention are suidatestrin and a modified form of thepseudo-oligosaccharide antibiotic validamycin (Asano N. et al., J.Antibiot. 40(4): 526-532, 1987; Goddijn O. J. et al., Plant Physiol.113(1): 181-190, 1997; Knuesel I. et al., Comp. Biochem. Physiol. B.Biochem. Mol. Biol. 120(4): 639-646, 1998). The validamycin must hereinbe modified such it can no longer enter the cell. Other compounds whichinhibit the activity of endogenous extracellular trehalase and which arenot taken up into the cell can however also be used according to theinvention.

The term “population of cells” is understood to mean according to theinvention a population of individual cells, as well as cells in tissuesand organs or parts thereof; or cells in whole organisms such as forinstance plants, wherein the whole plants or parts thereof can consistof the genetically transformed cells.

The method according to the invention is preferably used to selectgenetically modified plant cells. Seedlings of for instance Arabidopsiscannot develop further on media containing increased concentrations oftrehalose. While seeds will germinate, the formation of an extensiveroot system and the development of the first leaf stage are inhibited bythe presence of trehalose. Because the genetically transformed plantsare able to express a trehalase, particularly in the cytoplasm of thecell, due to the introduction of the selection-nucleotide sequence, thetrehalose which enters the cell can be broken down to glucose. Theglucose can then be used as nutrient source for the plant. Thegenetically transformed plants will therefore develop further, while thedevelopment of the non-transformed plants lags behind. When the methodaccording to the invention is used for the selection of geneticallytransformed cells in plants, the transformed plants can readily beidentified visually.

The method according to the present invention therefore provides asimple, environmentally-friendly selection system for transformed cells,particularly for genetically transformed plants. Trehalose is a simplecompound which is relatively inexpensive te produce and which hasmoreover been found to be non-toxic for humans and animals. Humans havethus been consuming large quantities of trehalose for a long time inproducts of yeast fermentation such as bread and beer, and humans andanimals are continually exposed to trehalose due to the presence oftrehalose-producing microbes in the intestinal flora.

According to the invention any nucleotide sequence which codes for aprotein with trehalase activity can be used as selection-nucleotidesequence in the genetically transformed cells. Use can for instance bemade of exogenous trehalase genes, such as for instance come frombacteria such as E. coli, although use can also be made of endogenoustrehalase genes, wherein the genes are modified such that they code formodified forms of the endogenous trehalase, for instance for anintracellular form of the normally only extracellularly activetrehalase. The advantage of using such endogenous trehalase genes isthat no additional foreign genetic material is introduced into the cell.

The desired transgene and the selection-nucleotide sequence can beintroduced into the cell for transforming using standardmolecular-biological techniques. Although this is not essential, thetransgene and selection gene can herein be linked to each other so thatthe presence of the selection gene always signifies that the transgeneis also present. The transgene and the selection gene can optionallyform part of the same genetic construct and be introduced via the samevector into the cell. In order to ensure that the selection-nucleotidesequence is expressed in the transformed cells, such a genetic constructwill further also comprise regulatory sequences such as for instance aconstitutive or regulatable promotor.

The method according to the invention can be used in particularlysuitable manner to select transgenic plants. Examples of plants forwhich the method according to the invention-can be used are for instancemaize (Zea mays L.), wheat (Triticum aestivm L.), barley (Hordeumvulgare L.), rice (Oryza sativa L.), soyabean (Phaseolus vulgaris L.),sugar beet (Beta vulgaris L.), chicory (Cichorum intybus L.), rapeseed(Brassica napus L.), sugar cane (Saccharum officinarum L.), sweet potato(Diocorea esculenta L.), manioc (Manihot esculenta L.), potato (Solanumtuberosum L.), tomato (lycopersicon esculentum L.) and grasses (forinstance Lolium ssp. Poa spp. and Festuca spp.).

The present invention further relates to the transformed cells which areselected using the method according to the invention, in particularplant cells, and to the plants regenerated therefrom, and their seedsand progeny.

The invention is further elucidated with reference to the accompanyingexamples and figures.

FIG. 1 shows the sensitivity of two different accessions of Arabidopsisthaliana to trehalose. A: seeds cultured in the presence of 100 mMmannitol (control); B: seeds cultured in the presence of 100 mMtrehalose.

FIG. 2 shows different constructs for cytoplasmic expression of the E.coli trehalase gene.

FIG. 3 shows a culture of transformed and non-transformed Arabidopsisthaliana Col.O seedlings in the presence of 100 mM trehalose.

FIG. 4 shows the nucleotide sequence of the TreF gene from E. coli.

FIG. 5 shows the mRNA sequence of Arabidopsis thaliana AtTre1.

FIG. 6 shows the mRNA sequence of GMTre1 from Glycine max.

EXAMPLES Example 1 Sensitivity to Trehalose of Seedlings of Arabidopsisthaliana Col. O and La-er

Seeds were sterilized using the gas-phase protocol of Clough and Bent(1998) (Clough S. J., Bent A. F., Plant J. 16(6): 735-743, 199.8) inEppendorf tubes. The sterilized sees were then resuspended in sterilewater and arranged on 0.8% w/v agar medium containing half-strengthMurashigue and Skoog medium (MS medium; Murashigue T, Skoog F, Physiol.Plant. 15: 473-497, 1977), vitamins and MES buffer (pH 5.7),supplemented with mannitol or trehalose in a final concentration of 100mM.

The seeds cultured in the presence of 100 mM mannitol developed intoplants with an extensive root system, while a lesser development wasobserved in the plants cultured in the presence of 100 mM trehalose, asshown in FIG. 1. FIG. 1A herein shows the seeds cultured in the presenceof 100 mM mannitol (control) and FIG. 1B the seeds cultured in thepresence of 100 mM trehalose.

The upper row of plants are the Arabidopsis thaliana Landsberg erecta(La-er), and the lower row of plants are the Arabidopsis thalianaColombia (Col.O).

Example 2 Expression Vectors with the E. coli Cytoplasmic Trehalase Gene(TreF) as Selection Gene

TreF was amplified from the genomic DNA of E. coli using the PCR-primersTreld (CTC TGC AGA TGC TCA ATC AGA AAA TTC AAA ACC) (SEQ ID NO:4) andTrelu (TGC ACT GCA GTT ATG GTT CGC CGT ACA AAC CAA) (SEQ ID NO:5). Theamplified TreF was then cloned in the pGEMT vector (Promega, US). TheTreF gene was further modified for the introduction of a myc-tag at theC-terminal end of the protein using the PCR primers TRe2d (AGC ACT GCAGCC ATG GCT TTG GTT ACC CTC AAT CAG AAA ATT CAA AAC CCT) (SEQ ID NO:6)and Tremyc (TTA CAG ATC TTC TTC AGA AAT AAG TTT TTG TTC TGG TTC GCC GTACAA ACC AAT TAA) (SEQ ID NO:7) and again cloned in pGEMT for sequencevalidation. The resulting modified TreF sequence was cut with Pst1restriction enzyme and introduced into:

A. pCAMBIA2201 (CAMBIA, Australia). The outer ends of the TreF fragmentwere blunted and the fragment ligated in pCAMBIA220 plasmid digested andblunted with NcoI and BstEII.

B. Pst1 site of pCambia 2380; for this purpose the ubiquitin 10 promotorof Arabidopsis thaliana was added as blunted Xho1-Spe 1 fragment in theblunted HindIII site of pcambia 2380-TreF.

C. Pst1 site of the pACN plasmid (Zeneca, Caddick M. X. et al., Nat.Biotechnol. 16(2): 177-180, 1998). The resulting construct was thendigested with HindIII, wherein a fragment was released having thereonthe hybrid AlcA/minimal 35S promotor, followed by the TreF sequence andthe Nos PolyA terminator. This fragment was inserted into the HindIIIsite of the SRN binary vector (Zeneca), whereby the shown construct wasobtained.

FIG. 2 shows the obtained constructs. LB: left T-DNA boundary; RB: rightT-DNA boundary; treF: E. coli gene coding for the cytoplasmic trehalase;NptII: neomycin phosphotransferase gene II: AlcR: gene coding forregulator of the alcohol inducable system; CaMV35S: cauliflower mosaicvirus 35S promotor; Ubi10: promotor of the ubiquitin 10 gene ofArabidopsis thaliana; AlcA/35S: AlcA promotor element reacting toethanol induction, fused with the CaMV35S promotor.

Example 3 Selection of Transgenic Arabidopsis Seedlings

Arabidopsis thaliana Col.O plants were transformed with Aarobacteriumwith the binary plasmid as described in Example 2C, via the “floral dip”protocol (Clough and Bent, supra). The obtained dry seeds weresterilized and sown on 0.8% w/v solid agar medium having thereinhalf-strength MS-salts (½ MS-salts), vitamins and MES buffer pH 5.7,supplemented with trehalose in a final concentration of 100 mM. Thedishes were incubated for 3 days at 4° C. (stratification), whereafter adrop of ethanol was applied to the inside of the cover and dishes weretransferred to 22° C.

FIG. 3 shows the seedlings obtained after 12 days. The transformedresistant seedlings are green, have long roots and primary leaves. Thenon-transformed, sensitive seedlings on the other hand accumulateanthocyanins, develop no primary leaves and the roots do not becomelonger than 3 mm. In moist conditions the cotyledons become pale.

Of the approximately 4000 sown plants, 24 resistant seedlings wereidentified. This shows that the transformation-frequency is comparableto that obtained with other selection systems. Twelve seedlings weretransferred to black earth and developed into plants which could not bedistinguished from non-transformed wild-type plants.

Example 4 Stable Expression of the Trehalase-Coding Selection Gene inArabidopsis Transgenic Lineages

The stability of the expression of the selection gene coding fortrehalase was tested in independent Arabidopsis transgenic lineagesusing the conventional kanamycin selection gene linked to the selectiongene according to the invention.

T2 seeds obtained from ten self-pollinated T1 plants from Example 3 weresterilized and sown on 0.8% solid agar medium having therein ½MS-salts,supplemented with 1% w/v sucrose and 25 mg/l kanamycin, incubated for 3days at 4° C., transferred to 22° C. and grown for 12 days in a 16-hourlight/8-hour dark cycle. The germination frequency was 100%.

Kanamycin-sensitive seedlings germinated but had blanched cotyledons, noroot development and no primary leaf stage. Kanamycin-resistantseedlings were green, developed primary leaves and roots.

As shown in table 1, the transgenic construct of the T1 plants wasalways passed on to the T2 generation. Table 1 shows the number ofseedlings resistant to kanamycin in each tested lineage.

Arabidopsis produces seeds through self-fertilization and is a diploidplant. When the T1 generation plant has at least one stable transgene inits genome, the T2 generation will consist of at least ¾ resistantplants and a maximum of ¼ sensitive plants. When the transgene is notinserted in stable manner in the genome of the T1 plants, the transgenewill not be found in the T2 generation. Table 1 shows that, by makinguse of kanamycin-selection on the T2 generation, T2 seedlings with thetransgene can be found in each T1 lineage.

TABLE 1 Ti kanamycin-sensitive kanamycin-resistant lineage seedlingsseedlings TF1 5 23 TF2 3 7 TF4 21 54 TF5 14 48 TF7 24 65 TF8 13 42 TF9 080 TF10 9 28 TF11 10 47 TF12 15 54

1. Method for selecting genetically transformed cells from a populationof cells, comprising of a) introducing into a cell at least one desirednucleotide sequence and at least one selection-nucleotide sequence toobtain a genetically transformed cell, wherein the selection-nucleotidesequence comprises a region which codes for a protein involved in themetabolizing of trehalose, wherein protein has trehalase activity; b)placing a population with transformed and non-transformed cells intocontact with trehalose and/or methylated or halogenated forms oftrehalose; and c) selecting the transformed cells from the population onthe basis of the capacity of the transformed cells to metabolize thetrehalose and/or methylated or halogenated forms of trehalose.
 2. Methodas claimed in claim 1, characterized in that the selection-nucleotidesequence comprises a modified endogenous trehalase gene which codes foran intracellularly active trehalase.
 3. Method as claimed in claim 1,characterized in that the selection-nucleotide sequence comprises theTreF gene from E. Coli.
 4. Method as claimed in claim 1, characterizedin that the selection-nucleotide sequence comprises the AtTRE1 gene fromArabidopsis.
 5. Method as claimed in claim 1, characterized in that themethod further comprises of: also bringing the population of cells intocontact with at least one inhibitor of endogenous extracellulartrehalase before or during step b).
 6. Method as claimed in claim 1,characterized in that the cell is a plant cell.
 7. Method for usingtrehalose and/or a methylated or halogenated forms of trehalose for theselection of transformed cells from a population of transformed andnon-transformed cells, wherein the genome of the transformed cellscomprises at least one selection-nucleotide sequence comprising a regionwhich codes for a protein involved in the metabolizing of trehalose andthe selection-nucleotide sequence comprises a region which codes for anintracellular protein with trehalase activity.
 8. Method according toclaim 7, characterized in that the selection-nucleotide sequencecomprises a modified endogenous trehalase gene which codes for anintracellularly active trehalase.
 9. Method according to claim 7,characterized in that the selection-nucleotide sequence comprises theTreF gene from E. Coli.
 10. Method according to claim 7, characterizedin that the selection-nucleotide sequence comprises the AtTRE1 gene fromArabidopsis.
 11. Method according to claim 7, characterized in that thecell is a plant cell.